Mucosal Immunity Of Avian Influenza Virus Antigen Induced By FcRn Mediated IgG Transport Pathway

The highly pathogenic avian influenza virus not only cause huge economic losses to poultry industry, but also pose a serious threat to human health.The avian influenza virus infect human mainly through mucosal surfaces, thus the most ideal way of preventing avian influenza is by mucosal immunization.Because subcutaneous or intramuscular immunization generally induce weak mucosal immune response, intranasal immunization can simulate the natural infection of Avian influenza virus. Mucosal vaccination not only can induce mucosal immune response at local mucosal sites, but also induce systemic immunity. As FcRn can efficiently transport IgG across the mucosal barrier,using mice as a model and designing a fusion of avian H5N1HA1to the IgG Fc fragment, we carry out the study of Avian influenza virus mucosal immune by FcRn mediated IgG transport pathway.In our previous studies, we have been constructed prokaryotic expression recom-binant plasmid of KG-HA1, pET-32a-HAl-wFc and insect cell eukaryotic expression recombinant plasmid of pFASTBAC HTb-sHAl-wFc and pFASTBAC HTb-sHAl-mFc. HA1-wFc was a fusion gene of HA1and the wild type fragments of IgG2a Fc, sHAl-wFc was a fusion gene of HA1which contain signal peptide and the wild type fragments IgG2a Fc,sHA1-mFc was a fusion gene of sHA1and the mutant fragment of IgG2a Fc.The main results are as follows:1. Four HA1fusion proteins were expressed and purified respectively:GST-HA1, His-HAl-wFc (prokaryotic expression), His-sHA1-wFc (eukaryotic expression, sHA1-wFc) and His-sHAl-mFc (eukaryotic expression, sHA1-mFc). Their molecular weight was62KDa,87KDa,72KDa and72KDa respectively.2. Using IMCD-rFcRn cell line as an in vitro transcytosis model, FcRn-dependent transcytosis of sHAl-wFc fusion protein applied to the apical reservoir was transported to the basolateral reservoir, but sHA1-mFc and His-HAl-wFc fusion proteins were not transported across the IMCD-rFcRn monolayer. sHA1-wFc fusion protein can effectively across the respiratory epithelium in mice and enter the blood in vivo.The difference was extremely significant compared to sHA1-mFc and GST-HA1fusion proteins (P<0.01).3.(1) On the10th day after the boost, SIgA antibody titers was1:117and1:64in the trachea and lungs washings of mice respectively from sHA1-wFc intranasal immunization group by ELISA, and IgG antibody titers was1:298and1:462respectively, the difference was extremely significant compared with other immunization groups (P<0.01).(2)On the28th day,the IgG antibody level of sHAl-wFc intranasal immunization group was the highest, ELISA antibody titer was1:9386, HI antibody titer was1:46,the difference was extremely significant compared with other intranasal immunization groups (P<0.01). However, the IgG antibody level of sHA1-wFc subcutaneous immunization group was also the highest. ELISA antibody titer was1:18773, HI antibody titer was1:80,the difference was extremely significant compared with sHA1-wFc intranasal immunization group (P<0.01).(3) On the10th day after the boost,the serum IFN-y and IL-2contents of sHAl-wFc intranasal immunization group was330pg/mL and106pg/mL respectively, the difference was extremely significant compared to other immunization groups (P <0.01).(4) On the10th day after the boost,the mice spleen cell stimulation index(SI) of sHAl-wFc intranasal immunization group was1.14, the difference was extremely significant compared with other intranasal immunization groups (P<0.01). The SI of sHA1-wFc subcutaneous immunization group was1.36, the difference was significant compared to sHAl-wFc intranasal immunization group (P<0.05).(5) On the3th day after a challenge, the hemagglutination activity of the virus in the lungs was not detected in sHAl-wFc immunization groups(intranasal or subcutaneous injection),while it could be detected in other immunization groups.The weight loss of mice in sHAl-wFc immunization groups (intranasal or subcutaneous injection) were not obvious, and it returned to normal weight soon, but the weight of mice in other immunization groups were lost significantly. The mice in sHAl-wFc immunization groups (intranasal or subcutaneous injection) were both100%protected against a viral challenge, while the survival rate of mice in sHAl-mFc and His-HAl-wFc intranasal immunization groups were40%and20%respectively.sHAl-wFc fusion protein can effectively across respiratory mucosa epithelial cells by FcRn mediated IgG transport pathway,with the help of adjuvant CpG, it could induce efficient mucosal and systemic immune responses and completely protect mice against a virus challenge.